Metabolic engineering of amino acid production

ABSTRACT

The present invention is directed towards the fermentative production of amino acids, providing microorganisms, methods and processes useful therefor. Microorganisms of the invention are capable of converting glucose to amino acids other than L-isoleucine, L-leucine and L-valine with greater efficiency than the parent strain. The efficiency of conversion may be quantified by the formula: [(g amino acid produced/g dextrose consumed)*100]=% Yield and expressed as yield from dextrose. The invention provides microorganisms that are made auxotrophic or bradytrophic for the synthesis of one or more branched chain amino acids by mutagenesis and selected for their ability to produce higher percent yields of the desired amino acid than the parental strain. Preferred microorganisms are  Corynebacterium, Brevibacterium  or  Escherichia coli  producing L-lysine. Mutagenesis is performed by classical techniques or through rDNA methodology. Methods of the invention are designed to increase the production of an amino acid by mutagenizing a parental strain, selecting cells auxotrophic or bradytrophic for the synthesis of one or more branched chain amino acids and selecting branched chain amino acid auxotrophs or bradytrophs that produce a higher percent yield from dextrose of the desired amino acid than the parental strain. Processes of the invention are designed for the production an amino acid comprising culturing in a medium a microorganism obtained by mutagenizing a parent strain to be auxotrophic or bradytrophic for branched chain amino acid synthesis and selecting variants that are capable of converting glucose to amino acids other than L-isoleucine, L-leucine and L-valine with greater efficiency than the parent strain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 11/320,139, filed Dec. 28, 2005, which is a continuation ofpending U.S. patent application Ser. No. 09/630,453, filed on Aug. 2,2000, now abandoned, which claimed priority to U.S. Provisional PatentApplication No. 60/146,379, filed on Aug. 2, 1999, now abandoned. Allpriority applications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the areas of microbial genetics and recombinantDNA technology. More specifically, the present invention relates to thefermentative production of amino acids. The invention providesmicroorganisms useful for the production of amino acids, methods toincrease the production of amino acids and processes for the productionof amino acids.

2. Related Art

The production of amino acids through fermentation enables inexpensiveproduction from cheap carbon sources such as molasses, acetic acid andethanol. Following the recognition that Corynebacteria were useful forthe industrial production of amino acids (S. Kinoshita et al.,Proceedings of the International Symposium on Enzyme Chemistry 2:464-468 (1957)), commercial production of amino acids by fermentativeprocesses was made more possible with the isolation of mutant strains.Microorganisms employed in microbial processes for amino acid productionmay be divided into 4 classes: wild-type strain, auxotrophic mutant,regulatory mutant and auxotrophic regulatory mutant (K. Nakayama et al,in NUTRITIONAL IMPROVEMENT OF FOOD AND FEED PROTEINS, M. Friedman, ed.,(1978), pp. 649-661). The stereospecificity of the amino acids producedby fermentation makes the process advantageous compared with syntheticprocesses; amino acids produced by microbial process are generally theL-form.

L-lysine is one example of an amino acid produced by industrialfermentation. Commercial production of this essential amino acid isprincipally done utilizing the gram positive Corynebacterium glutamicum,Brevibacteriumflavum and Brevibacterium lactofermentum (Kleemann, A.,et. al., Amino Acids, in ULLMAN'S ENCYCLOPEDIA of INDUSTRIAL CHEMISTRY,vol. A2, pp. 57-97, Weinham: VCH-Verlagsgesellschaft (1985));cumulatively, these three organisms presently account for theapproximately 250,000 tons of L-lysine produced annually.

Given the economic importance of L-lysine production by fermentiveprocesses, it would be beneficial to increase the total amount producedwhile simultaneously decreasing production costs. To that end, thebiochemical pathway for L-lysine synthesis has been intensivelyinvestigated in Corynebacterium (recently reviewed by Sahm et al., Ann.N.Y. Acad. Sci. 782: 25-39 (1996)). Entry into the lysine pathway beginswith L-aspartate (see FIG. 1), which itself is produced bytransamination of oxaloacetate. A special feature of C. glutamicum isits ability to convert the lysine intermediate piperideine2,6-dicarboxylate to diaminopimelate by two different routes, i.e. byreactions involving succinylated intermediates or by the single reactionof diaminopimelate dehydrogenase. Overall, carbon flux into the pathwayis regulated at two points: first, through feedback inhibition ofaspartate kinase by the levels of both L-threonine and L-lysine; andsecond through the control of the level of dehydrodipicolinate synthase.Therefore, increased production of L-lysine may be obtained inCorynebacteria by deregulating and increasing the activity of these twoenzymes.

In addition to the biochemical pathway leading to L-lysine synthesis,recent evidence indicates that consideration of lysine transport out ofcells into the media is another condition to be considered in thedevelopment of lysine over-producing strains of C. glutamicum. Studiesby Krämer and colleagues indicate that passive transport out of thecell, as the result of a leaky membrane, is not the sole explanation forlysine efflux; their data suggest a specific carder with the followingproperties: (1) the transporter possesses a rather high Km value forlysine (20 mM); (2) the transporter is an OH⁻ symport system (uptakesystems are H+ antiport systems); and (3) the transporter is positivelycharged, and membrane potential stimulates secretion (S. Bröer and R.Krämer, Eur. J. Biochem. 202: 137-143 (1991).

Several fermentation processes utilizing various strains isolated forauxotrophic or resistance properties are known in the art for theproduction of L-lysine: U.S. Pat. No. 2,979,439 discloses mutantsrequiring homoserine (or methionine and threonine); U.S. Pat. No.3,700,557 discloses mutants having a nutritional requirement forthreonine, methionine, arginine, histidine, leucine, isoleucine,phenylalanine, cystine, or cysteine; U.S. Pat. No. 3,707,441 discloses amutant having a resistance to a lysine analog; U.S. Pat. No. 3,687,810discloses a mutant having both an ability to produce L-lysine and aresistance to bacitracin, penicillin G or polymyxin; U.S. Pat. No.3,708,395 discloses mutants having a nutritional requirement forhomoserine, threonine, threonine and methionine, leucine, isoleucine ormixtures thereof and a resistance to lysine, threonine, isoleucine oranalogs thereof; U.S. Pat. No. 3,825,472 discloses a mutant having aresistance to a lysine analog; U.S. Pat. No. 4,169,763 discloses mutantstrains of Corynebacterium that produce L-lysine and are resistant to atleast one of aspartic analogs and sulfa drugs; U.S. Pat. No. 5,846,790discloses a mutant strain able to produce L-glutamic acid and L-lysinein the absence of any biotin action-surpressing agent; and U.S. Pat. No.5,650,304 discloses a strain belonging to the genus Corynebacterium orBrevibacterium for the production of L-lysine that is resistant to4-N-(D-alanyl)-2,4-diamino-2,4-dideoxy-L-arabinose2,4-dideoxy-L-arabinose or a derivative thereof.

More recent developments in the area of L-lysine fermentive productionin Corynebacteria involve the use of molecular biology techniques toaugment lysine production. The following examples are provided as beingexemplary of the art: U.S. Pat. Nos. 4,560,654 and 5,236,831 disclose anL-lysine producing mutant strain obtained by transforming a hostCorynebacterium or Brevibacterium microorganism which is sensitive toS-(2-aminoethyl)-cysteine with a recombinant DNA molecule wherein a DNAfragment conferring both resistance to S-(2-aminoethyl)-cysteine andlysine producing ability is inserted into a vector DNA; U.S. Pat. No.5,766,925 discloses a mutant strain produced by integrating a genecoding for aspartokinase, originating from coryneform bacteria, withdesensitized feedback inhibition by L-lysine and L-threonine, intochromosomal DNA of a Coryneform bacterium harboring leaky typehomoserine dehydrogenase or a Coryneform bacterium deficient inhomoserine dehydrogenase gene.

In addition to L-lysine, Corynebacterium and related organisms areuseful for the production of other amino acids, for example the branchedchain amino acids L-leucine, L-isoleucine and L-valine. The biochemicalpathways leading to branched chain amino acid biosynthesis are also wellstudied. Carbon flux into the aspartate pathway may be funneled onto theproduction of L-lysine or L-threonine, which may be utilized for theproduction of L-isoleucine (FIG. 1B). L-isoleucine is produced fromL-threonine in five reactions; the enzymes catalyzing these reactionsinclude: (1) threonine dehydratase; (2) acetohydroxy acid synthase; (3)isomeroreductase; (4) dihydroxy acid dehydratase; and (5) transaminaseB. Threonine dehydratase is the only enzyme in this pathway unique toisoleucine synthesis; the other four enzymes are also utilized in theproduction of the other branched chain amino acids, valine and leucine.Carbon flux from threonine to isoleucine is controlled by threoninedehydratase and acetohydroxy acid synthase (AHAS). With the cloning ofgenes encoding the enzymes of the isoleucine pathway (ilvA, ilvB, ilvC,ilvD and ilvE) in Corynebacterium (C. Cordes et al., Gene 112:113-116 (I992); B. Möckel et al., J. Bacteriology 174: 8065-8072 (1992); and C.Keilhauer et al., J. Bacteriology 175: 5595-5603 (1993)), recombinantDNA techniques may be applied to generate novel strains.

Improvements in the production of the amino acids L-isoleucine,L-leucine and L-valine by increasing the activity of enzymes in thebranched chain amino acid biosynthetic pathway have been described.Additionally, improvements in the production of branched chain aminoacids by improving the acetohydroxy acid synthase (AHAS) activityencoded by the ilvBN operon have been described. (see generally H. Sahmet al., Ann. N.Y. Acad. Sci. 782:25-39 (1996)).

Exemplary processes for the production of branched chain amino acidsinclude the following: U.S. Pat. No. 5,188,948 discloses a fermentationprocess for producing L-valine utilizing a microorganism is resistant toa polyketide; U.S. Pat. No. 5,521,074 discloses a process for producingL-valine utilizing a microorganism which belongs to the genusCorynebacterium or Brevibacterium, which exhibits a) an ability toproduce L-valine, b) resistance to L-valine in a medium containingacetic acid as a sole carbon source, and c) sensitivity to a pyruvicacid analog in a medium containing glucose as a sole carbon source; U.S.Pat. No. 4,601,983 discloses a genetic sequence coding for theproduction of a protein having the activity of homoserine dehydrogenasecapable of replication in coryneform bacteria and used to produceL-threonine and L-isoleucine; U.S. Pat. No. 4,442,208 discloses afermentation process for the production of L-isoleucine utilizing aBrevibacteriumor Corynebacterium strain obtained by recombinant DNAtechniques that is resistant to α-amino-β-hydroxy valeric acid; U.S.Pat. No. 4,656,135 discloses a process for producing L-isoleucine, whichcomprises culturing a microorganism belonging to the genusBrevibacterium or the genus Corynebacterium which has a methyllysineresistance or α-ketomalonic acid resistance and which is capable ofproducing L-isoleucine in a liquid medium, and obtaining the accumulatedL-isoleucine from said medium; U.S. Pat. No. 5,118,619 discloses amethod for the fermentative production of L-isoleucine fromD,L-α-hydroxybutyrate by means of mutants that utilize D-lactate; U.S.Pat. No. 5,763,231 discloses a process for producing L-leucine, whichincludes incubating a strain of the genus Corynebacterium, Escherichia,Brevibacterium, or Microbacterium in a culture medium and reacting theresulting cells with saccharides and acetic acid or its salt to form andaccumulate L-leucine in the reaction solution; and U.S. Pat. No.3,970,519 discloses strains that resist feedback inhibition by leucineor its analogs and require at least one of isoleucine, threonine ormethionine as a growth nutriment to produce L-leucine.

Improvements in the production of amino acids by decreasing theproduction of valine have not been described.

Improvements in the production of amino acids by decreasing AHASactivity have not been described.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide microorganisms thatare capable of converting glucose to amino acids other thanL-isoleucine, L-leucine and L-valine with greater efficiency than theparent strain. The efficiency of conversion may be quantified by theformula:

[(g amino acid produced/g dextrose consumed)*100]=% Yield and expressedas yield from dextrose.

In one embodiment, the invention provides microorganisms that are madeauxotrophic for the synthesis of one or more branched chain amino acidsby mutagenesis arid selected for their ability to produce higher percentyields of the desired amino acid than the parental strain

In a more specific embodiment of the invention provides microorganismsobtained by subjecting a parental strain to random chemical mutagenesis,isolating a mutagenized variant that is auxotrophic for branched chainamino acid synthesis and selecting variants that are capable ofconverting glucose to amino acids other than L-isoleucine, L-leucine andL-valine with greater efficiency than the parent strain. Anotherspecific embodiment of the invention provides microorganisms obtained byutilizing rDNA methodologies to introduce a change (i.e., a mutation) inthe nucleic acid sequence of the ilvBN operon, isolating a mutagenizedvariant that is auxotrophic or bradytrophic for branched chain aminoacid synthesis and selecting variants that are capable of convertingglucose to amino acids other than L-isoleucine, L-leucine and L-valinewith greater efficiency than the parent strain.

In a preferred embodiment, the microorganisms of the invention produceL-lysine. Another preferred embodiment of the invention is drawn toCorynebacterium microorganisms, or Brevibacteriummicroorganisms, andparticularly preferred microorganisms are Corynebacterium orBrevibacteriummicroorganisms that produce L-lysine. In a most preferredembodiment, the microorganisms have the identifying characteristics ofNRRL No. B-30149 (also known as LC10) or NRRL No. B-30150 (also known asBF100-1030), strains deposited on Jun. 29, 1999 with the AgriculturalResearch Service Culture Collection (NRRL), 1815 North UniversityStreet, Peoria, Ill. 61604 USA.

Another object of the invention provides methods to increase theproduction of an amino acid by mutagenizing a parental strain, selectingcells auxotrophic for the synthesis of one or more branched chain aminoacids and selecting branched chain amino acid auxotrophs that produce ahigher percent yield from dextrose of the desired amino acid than theparental strain.

In a preferred embodiment, the method is drawn to increasing the yieldfrom dextrose of the amino acid L-lysine obtained by culturingCorynebacterium which, through random chemical mutagenesis orrecombinant DNA (rDNA) technology, is made to be auxotrophic orbradytrophic for one or more of the branched chain amino acids leucine,isoleucine and valine.

In one specific embodiment, branched chain amino acid auxotrophy is theresult of chemical mutagenesis of Corynebacterium. In an alternativespecific embodiment, branched chain amino acid auxotrophy is the resultof mutagenesis of the ilvBN operon by rDNA techniques.

Another object of the invention is to provide processes for theproduction of an amino acid from microorganisms that are capable ofconverting glucose to amino acids other than L-isoleucine, L-leucine andL-valine with greater efficiency than the parent strain.

In one embodiment, the invention provides a process for producing anamino acid comprising culturing in a medium a microorganism obtained bymutagenizing a parent strain to be auxotrophic or bradytrophic forbranched chain amino acid synthesis and selecting variants that arecapable of converting glucose to amino acids other than L-isoleucine,L-leucine and L-valine with greater efficiency than the parent strain.

In a preferred embodiment for the process, the microorganism utilized infermentation is obtained by subjecting the parent strain to randomchemical mutaganesis, isolating a mutagenized variant that isauxotropkic for branched chain amino acid synthesis and selectingvariants that are capable of converting glucose to amino acids otherthan L-isgleucine, L-leucine and L-valine with greater efficiency thanthe parent strain. In another preferred embodiment for the process, themicroorganism utilized in fermentation is obtained by altering (i.e.,introducing a mutation) the nucleotide sequence of the ilvBN operon byrDNA methodology, isolating a mutagenized variant that is auxotrophic orbradytrophic for branched chain amino acid synthesis and selectingvariants that are capable of converting glucose to amino acids otherthan L-isoleucine, L-leucine and L-valine with greater efficiency thanthe parent strain.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) A schematic presentation of the biochemical pathway leadingto L-lysine production in Corynebacterium; (B) A schematic presentationof the biochemical pathway leading to L-isoleucine production inCorynebacterium.

FIG. 2. A-B) Presentation of the nucleotide sequence of the ilvBN operonof Corynebacterium (SEQ ID NO:1); C) Presentation of the amino acidsequence of the ilvBN operon of Corynebacterium (SEQ ID NO:2).

FIG. 3. A-B) Presentation of the nucleotide sequence for the ilvBNdeletion mutant in the plasmid pAL203Δ (SEQ ID NO:3); C) Presentation ofthe amino acid sequence for the ilvBN deletion mutant in the plasmidpAL203Δ (SEQ ID NO:4).

FIG. 4. A-C) Presentation of the nucleotide sequence of the pRV1B5allele (SEQ ID NO:5); D) Presentation of the amino acid sequence of thepRV1B5 allele (SEQ ID NO:6).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Definitions

In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

Auxotroph: As used herein, the term auxotroph refers to a strain ofmicroorganism requiring for growth an external source of a specificmetabolite that cannot be synthesized because of an acquired geneticdefect.

Amino Acid Supplement: As used herein, the term “Amino Acid Supplement”refers to an amino acid required for growth and added to minimal mediato support auxotroph growth.

Bradytroph: As used herein, the term bradytroph refers to a strain ofmicroorganism that exhibits retarded growth in the absence of anexternal source of a specific metabolite. A bradytroph can synthesizethe metabolite, but because of an acquired genetic defect, the rate ofsynthesis is less than the parent strain's rate of synthesis of the samemetabolite.

Branched Amino Acid: As used herein, the term refers to those aminoacids in which the R group possesses a branched carbon structure, suchas leucine, isoleucine and valine.

Carbon Flux: As used herein, the term refers to the movement of carbonbetween amphibolic, catabolic and/or anabolic biochemical pathways of anorganism.

Chromosomal Integration: As used herein, the term refers to theinsertion of art exogeneous DNA fragment into the chromosome of a hostorganism; more particularly, the term is used to refer to homologousrecombination between an exogenous DNA fragment and the appropriateregion of the host cell chromosome.

High Yield Derivative: As used herein, the term refers to strain ofmicroorganism that produces a higher yield from dextrose of a specificamino acid when compared with the parental strain from which it isderived.

Mutation: As used herein, the term refers to a single base pair change,insertion or deletion in the nucleotide sequence of interest.

Operon: As used herein, the term refers to a unit of bacterial geneexpression and regulation, including the structural genes and regulatoryelements, in DNA. Examples of regulatory elements that are encompassedwithin the operon include, but are not limited to, promoters andoperators.

Parental Strain: As used herein, the term refers to a strain ofmicroorganism subjected to some form of mutagenesis to yield themicroorganism of the invention.

Percent Yield From Dextrose: As used herein, the term refers to theyield of amino acid from dextrose defined by the formula [(g amino acidproduced/g dextrose consumed)*100]=% Yield.

Phenotype: As used herein, the term refers to observable physicalcharacteristics dependent upon the genetic constitution of amicroorganism.

Relative Growth: As used herein, the term refers to a measurementproviding an assessment of growth by directly comparing growth of aparental strain with that of a progeny strain over a defined time periodand with a defined medium.

Mutagenesis: As used herein, the term refers to a process whereby amutation is generated in DNA. With “random” mutatgenesis, the exact siteof mutation is not predictable, occurring anywhere in the genome of themicroorganism, and the mutation is brought about as a result of physicaldamage caused by agents such as radiation or chemical treatment, rDNAmutagenesis is directed to a cloned DNA of interest, and it may berandom or site-directed.

2. Microorganisms of the Invention Based on Decreased Carbon Flow toBranched Chain Amino Acid Synthesis and Increased Production ofNonBranched Amino Acids

The invention provides generally for the creation of microorganisms thatare auxotrophic for the branched chain amino acid synthesis in order todirect carbon flux to non-branched chain amino acid synthesis. Morespecifically, by selecting for a specific auxotrophic phenotyperequiring one or more of the branched chain amino acids leucine,isoleucine or valine (e.g., isoleucine and valine) or designingmutations in the ilvBN operon that decrease the flow of carbon toisoleucine, leucine and valine synthesis, carbon flux in the system maythen become available for other metabolic pathways (e.g, L-lysinesynthesis).

In one specific embodiment, the invention provides a microorganism Cthat produces amino acid X, wherein said microorganism C is obtained bythe following method:

-   -   (a) selecting a parental microorganism A that produces said        amino acid from dextrose in percent yield Y;    -   (b) mutagenizing said parental microorganism A to produce        microorganism B by a method selected from the group consisting        of:        -   (i) random chemical mutagenesis; and        -   (ii) rDNA mutagenesis of the ilvBN operon;    -   (c) selecting from step (b) at least one mutagenized        microorganism B that is auxotrophic or bradytrophic for one or        more of the branched chain amino acids leucine, isoleucine and        valine; and    -   (d) selecting from step (c) at least one microorganism C which        produces said amino acid X from dextrose in percent yield Z,        wherein said percent yield Z is greater than said percent yield        Y.        The percent yield from dextrose is preferably calculated using        the formula [(g amino acid/L/(g dextrose consumed/L)]*100.

Parental microorganisms may be selected from any microorganism known inthe art that can produce amino acid X. Particularly favored parentalmicroorganisms Corynebacterium and Brevibacterium.

The strains of the invention may be prepared by any of the methods andtechniques known and available to those skilled in the art. Illustrativeexamples of suitable methods for constructing the inventive bacterialstrains include but are not limited to the following: mutagenesis usingsuitable agents such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG); geneintegration techniques, mediated by transforming linear DNA fragmentsand homologous recombination; and transduction mediated by abacteriophage. These methods are well known in the art and aredescribed, for example, in J. H. Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); M. Singer andP. Berg, Genes & Genomes, University Science Books, Mill Valley, Calif.(1991); J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989); P. B. Kaufman et al., Handbook of Molecularand Cellular Methods in Biology and Medicine, CRC Press, Boca Raton,Fla. (1995); Methods in Plant Molecular Biology and Biotechnology, B. R.Glick and J. E. Thompson, eds., CRC Press, Boca Raton, Fla. (1993); andP. F. Smith-Keary, Molecular Genetics of Escherichia coli, The GuilfordPress, New York, N.Y. (1989).

A. Construction of Branched Chain Amino Acid Auxotrophs by RandomMutagenesis

One specific preferred embodiment of the invention provides thatmodification of an enzymatic step common to L-isoleucine, L-leucine andL-valine biosynthesis can increase the percent yield of L-lysine fromdextrose.

In a most preferred embodiment, the invention provides for theproduction of microorganisms that are auxotrophic for branched chainamino acid synthesis by means of random mutagenesis of a parental strainfollowed by selection of the specific phenotype. The parental strainchosen for mutagenesis may be any strain known to produce the amino acidof interest. Preferred organisms include Corynebacterium strains andBrevibacterium strains, and most preferred organisms includeCorynebacterium strains and Brevibacterium strains that produceL-lysine.

The parental strain may be mutagenized using any random mutagenesistechnique known in the art, including, but not limited to, radiation andchemical procedures. Particularly preferred is random chemicalmutagenesis, and most preferable is the alkylating agent methoddescribed by J. H. Miller (J. H. Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory (1972).

By way of example, chemical mutagenesis was conducted as follows. Aculture of lysine-producing Corynebacterium strain was grown in richmedium at 30° C. up to an optical density of 6.0. Cells were washed withminimal medium and resuspended in minimal medium containing 100micrograms per mL of NTG. Ceils were exposed to the mutagen for 30minutes at 30° C. Ceils were washed with minimal medium and plated ontorich medium. Colonies from rich medium were replica-plated to rich andminimal medium. Colonies that grew on rich medium but did not grow onminimal medium were classified as auxotrophs. Auxotroph mutants werereplica-plated onto minimal medium and minimal medium containing 10 mML-isoleucine and 10 mM L-valine. Colonies that were rescued by theisoleucine and valine were classified as valine auxotrophs. Strain B4Bis a valine auxotroph generated by chemical mutagenesis.

B. Construction of Branched Chain Amino Acid Auxotrophs by MutagenesisThrough rDNA Methodology

Another specific preferred embodiment of the invention utilizesrecombinant DNA technology to effect in vitro and in vivo mutagenesis ofcloned DNA sequences that encode proteins important for the biosynthesisof branched chain amino acids. The mutated DNA may then be used tomodify the parented strain to produce mutant strains that areauxotrophic for branched chain amino acid synthesis and that produce ahigher yield from dextrose of non-branched chain amino acids than theparental strain.

In one specific preferred embodiment, the cloned DNA of interest may bemutated through recombinant DNA technology by any means known in thethat art. As one skilled in the art would know, the mutations in thecloned DNA may constitute single nucleotide changes (point mutations),multiple nucleotide changes, nucleotide deletions or insertions. Generalmethods for recombinant DNA technology are known to those skilled in theart and may be found in a number of common laboratory manuals thatdescribe fundamental techniques, such as nucleic acid purification,restriction enzyme digestion, ligation, gene cloning, gene sequencing,polymerase chain amplification (PCR) of gene sequences, and the like.(see e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989); Current Protocols in Molecular Biology. Ausubel et al. eds.,John Wiley & Sons, New York, (1994); PCR Protocols, Innis et al., eds.,Academic Press, Inc., New York, pp. 407-415 (1990)).

In addition, references that specifically teach in vitro mutatgenesis ofcloned DNA are known to those skilled in the art. For example,strategies such as site-directed mutatgenesis, oligonucleotide-directedmutatgenesis, linker scanning mutatgenesis, random chemical mutatgenesisin vitro, cassette mutatgenesis, PCR mutatgenesis and others aredetailed in Directed Mutagenesis: A Practical Approach, M. J. McPherson,ed., Oxford University Press, New York, (1991).

In another specific preferred embodiment, the cloned DNA of interest maybe mutated in vivo in a host ceil. This type of “in vivo mutagenesis”includes processes of generating random mutations in any cloned DNA ofinterest by the propagation of the DNA in a strain of E. coli thatcarries mutations in one or more of the DNA repair pathways. These“mutator” strains have a higher random mutation rate than that of awild-type parent. Propagating the DNA in one of these strains willgenerate random mutations within the DNA. Systems designed to accomplishthis are known to those skilled in the art and are availablecommercially. For example, Stratagene, Inc. provides a system utilizingthe XL1 Red Strain of E. coli which has had its DNA repair genes (MutH,MutL and MutS) deleted such that many different mutations occur in ashort time. Up to 10,000 mutations may take place within a 30 hour timespan such that an entire mutated DNA library may be prepared frommutated DNA by procedures known in the art.

The cloned DNA selected for mutation may be any sequence known in theart to be important for branched chain amino acid synthesis, includingbut not limited to, sequences encoding one or more enzymes important forsynthesis or one or more protein products important for transport andexcretion. Most preferred is the cloned sequence for the ilvBN operon ofCorynebacterium or Brevibacterium. Corynebacterium genes involved inbranched chain amino acid synthesis have been cloned; for example, genesequences are available for the isoleucine pathway (ilvA, itvB, ilvC,ilvD and ilvE) (C. Cordes et al., Gene 112: 113-116 (1992); B. Möckel etal., J. Bacteriology 174: 8065-8072 (1992); and C. Keilhauer et al., J.Bacteriology 175: 5595-5603 (1993)).

L-isoleucine is produced from L-threonine in five reactions; the enzymescatalyzing these reactions include: (1) threonine dehydratase (ilvA);(2) acetohydroxy acid synthase (ilvBN); (3) isomeroreductase (ilvC); (4)dihydroxy acid dehydratase (ilvD); and (5) transaminase B (ilvE).Threonine dehydratase is the only enzyme in this pathway unique toisoleucine synthesis; the other four enzymes are also utilized in theproduction of the other branched chain amino acids, valine and leucine.The enzymatic pathway involved in isoleucine biosynthesis inCorynebacterium strains is presented in FIG. 2.

Acetohydroxy acid synthase (AHAS) and isomeroreductase (IR) catalyzesubsequent reactions in the flux of metabolites towards isoleucine,valine, leucine, and pantothenate. As in other bacteria, the AHAS ofCorynebacterium strains is encoded by two genes, ilvB and ilvN. Genedisruption verified that these genes encode the single AHAS activity inC. glutamicum (Keilhauer, C., et al., J. Bacteriology 175:5595-5603(1973)). Three transcripts of 3.9, 2.3, and 1.1 kb were identified invivo by Northern Blot analysis, which correspond to ilvBNC, ilvNC, andilvC messages, respectively. The ilvC transcript (encoding IR) is themost abundant transcript from the ilv operon of C. glutamicum.Additional analysis indicates that three promoters are active in thisoperon; the steady-state levels of the ilvBNC and ilvNC messagescontribute significantly to the total activity of the single AHAS.

In a most preferred invention embodiment, a mutation may be generated byway of restriction enzyme digestion to create a deletion in the clonedilvBN operon DNA sequence. The mutated ilvBN sequence may then besubstituted for the wild type sequence by homologous recombination andscreened for branched chain amino acid auxotrophy.

Another embodiment of the invention is drawn to a microorganismCorynebacterium having the following that is auxotrophic for the one ormore of the branched chain amino acids isoleucine, leucine and valineand produces a percent yield from dextrose of the an amino acid ofinterest that is greater than the parental strain percent yield. In aparticularly favored embodiment, the amino acid produced is L-lysine.

Other highly preferred embodiments of the invention are drawn tomicroorganisms having substantially all of the characteristics of NRRLDeposit No. B-30149, which was deposited on Jun. 29, 1999, with theAgricultural Research Culture Collection (1815 N. University Street,Peoria, Ill. 61604, USA) or NRRL Deposit No. B-30150, which wasdeposited on Jun. 29, 1999, with the Agricultural Research CultureCollection (1815 N. University Street, Peoria, Ill. 61604, USA). NRRLDeposit No. B-30149 is of the microorganism Corynebacterium glutamicumLC10, and NRRL Deposit No. B-30150 is of the microorganismCorynebacterium glutamicum BF100-1030.

3. Methods of Increasing the Production of an Amino Acid

A further object of the invention provides methods to increase theproduction of an amino acid. The invention provides generally for amethod to increase the production of an amino acid X, comprising:

-   -   (a) selecting a parental microorganism A that produces said        amino acid from dextrose in percent yield Y;    -   (b) mutagenizing said parental microorganism A to produce        microorganism B by a method selected from the group consisting        of:        -   (i) random chemical mutagenesis; and        -   (ii) rDNA mutagenesis of the ilvBN operon;    -   (c) selecting from step (b) at least one mutagenized        microorganism B that is auxotrophic for one or more of the        branched chain amino acids leucine, isoleucine and valine; and    -   (d) selecting from step (c) at least one microorganism C which        produces said amino acid X from dextrose in percent yield Z,        wherein said percent yield Z is greater than said percent yield        Y.

In one particular preferred embodiment, any strain known in the art maybe selected as a parental strain that produces the amino acid ofinterest at a determined percent yield from dextrose. The percent yieldfrom dextrose may be easily calculated using the following formula: [(gamino acid/L/(g dextrose consumed/L)]*100.

After selecting the organism and determining the percent yield fromdextrose of the amino acid, the microorganism is preferably subjected tomutagenesis either by random mutagenesis techniques directed at theentire genome of the organism or by rDNA techniques directed towardscloned DNA of interest. Regardless of the particular method ofmutagenesis employed, mutated organisms are screened and selected on thebasis of auxotrophy for branched chain amino acid synthesis. Auxotrophsselected may then be screened to determine which strains produce ahigher percent yield of the desired amino acid from dextrose than theparental strain.

Various embodiments of the invention include methods to increase theproduction of an amino acid of interest from the organismsCorynebacterium, Brevibacterium, and E. coli. Additionally, dependingupon the particular embodiment, the invention provides methods toincrease the production of non-branched amino acids, such as glycine;alanine; methionine; phenylalanine; tryptophan; proline; serine;threonine; cysteine; tyrosine; asparagine; glutamine; aspartic acid;glutamic acid; lysine; arginine; and histidine.

In a favored embodiment, the invention provides methods to increasenon-branched chain amino acid production by creating auxotrophs forbranched chain amino acid synthesis and diverting carbon flux from thesynthesis of leucine, isoleucine and valine. A particularly favoredembodiment is drawn to a method of increasing the production of an aminoacid by selecting from a mutagenized parental strain a strain that isauxotrophic for valine and isoleucine synthesis.

The invention further provides various preferred embodiments for methodsto increase the production of an amino acid wherein the parental strainmay be mutagenized either by random mutagenesis techniques (e.g.,radiation or chemical mutagenesis) or mutagenesis of the ilvBN operon byrDNA techniques. In one particular preferred embodiment, the parentalstrain may be mutagenized by random chemical mutagenesis. In anotherparticular preferred embodiment, the parental strain is mutagenized byrDNA techniques directed at the cloned ilvBN operon nucleotide sequence.

4. Processes for the Production of an Amino Acid

A further object of the invention provides processes for the productionof an amino acid. The invention provides generally for a process forproducing an amino acid X comprising:

-   -   (a) culturing a microorganism C in a medium, wherein said        microorganism C is obtained by the following method:        -   (i) selecting a parental microorganism A that produces said            amino acid from dextrose in percent yield Y;        -   (ii) mutagenizing said parental microorganism A to produce            microorganism B by a method selected from the group            consisting of:        -   (a) random chemical mutagenesis; and        -   (b) rDNA mutagenesis of the ilvBN open;        -   (iii) selecting from step (b) at least one mutagenized            microorganism B that is auxotrophic for one or more of the            branched chain amino acids leucine, isoleucine and valine;            and        -   (iv) selecting from step (c) at least one microorganism C            which produces said amino acid X from dextrose in percent            yield Z, wherein said percent yield Z is greater than said            percent yield Y; and    -   (b) recovering said amino acid X that is produced from said        microorganism C.

Preferred embodiments of the invention are drawn to processes in whichthe cultured microorganism is selected from the group that includesCorynebacterium, Brevibacterium, and E. coli. Particularly preferred areprocess drawn to the organisms of the genus Corynebacterium.Microorganisms selected for the processes of the invention are thosethat produce an amino acid of interest, particularly non-branched chainamino acids. More particularly preferred microorganisms aremicroorganisms that produce glycine; alanine; methionine; phenylalanine;tryptophan; proline; serine; threonine; cysteine; tyrosine; asparagine;glutamine; aspartic acid; glutamic acid; lysine; arginine; andhistidine. The level of production of the amino acid of choice mayconveniently determined by the following formula to calculate thepercent yield from dextrose: [(g amino acid/L/(g dextroseconsumed/L)]*100.

Microorganisms used in the processes of the invention are preferablyobtained by mutagenesis of the chosen parental strain. Preferredembodiments of the invention include processes in which the chosenparental strains are subjected either to random mutagenesis directed atthe entire genome or to rDNA mutagenesis of cloned DNA of interest.

Particularly preferred embodiments of the invention wherein the parentalstrain is subjected to random mutagenesis include but are not limitedto, mutagenesis by radiation treatment or chemical treatment. A moreparticularly preferred embodiment is drawn to random chemicalmutagenesis of the parental strain.

Another particularly preferred embodiment of the invention provides forrDNA mutagenesis of the parental strain. A more particularly preferredembodiment is drawn to rDNA mutagenesis of the ilvBN operon in vitro orin vivo. Mutated forms of the ilvBN operon, or fragments thereof, maythen be substituted for wild-type ilvBN operon sequence throughhomologous recombination techniques that are well known to those skilledin the art (see Example 6).

However the selected parental strains or cloned DNA sequences aremutagenized, the resultant progeny are screened and selected forauxotrophy for branched chain amino acid synthesis (i.e., leucine,isoleucine or valine). The selection of such mutants is well with in theskill of those in the art. A particularly preferred embodiment is drawnto strains that are auxotrophic for valine and isoleucine biosynthesis.

Ultimately, selection of the microorganisms of the processes of theinvention is dependent upon production of the amino acid of choice.Utilizing the formula [(g amino acid]L/(g dextrose consumed/L)]*100 todetermine the percent yield from dextrose, the desired microorganismsare selected on the basis of having a higher percent yield from dextroseof the amino acid of choice than the parental strain.

Other embodiments of the invention are drawn to processes that vary byway of the specific method of culturing the microorganisms of theinvention. Thus, a variety of fermentation techniques are known in theart which may be employed in processes of the invention drawn to theproduction of amino acids.

Illustrative examples of suitable carbon sources include, but are notlimited to: carbohydrates, such as glucose, fructose, sucrose, starchhydrulysate, cellulose hydrolysate and molasses; organic acids, such asacetic acid, propionic acid, formic acid, malic acid, citric acid, andfumaric acid; and alcohols, such as glycerol.

Illustrative examples of suitable nitrogen sources include, but are notlimited to: ammonia, including ammonia gas and aqueous ammonia; ammoniumsalts of inorganic or organic acids, such as ammonium chloride, ammoniumphosphate, ammonium sulfate and ammonium acetate; and othernitrogen-containing, including meat extract, peptone, corn steep liquor,casein hydrolysate, soybean cake hydrolysate and yeast extract.

Generally, amino acids may be commercially produced from the inventionin fermentation processes such as the batch type or of the fed-batchtype. In batch type fermentations, all nutrients are added at thebeginning of the fermentation. In fed-batch or extended fed-batch typefermentations one or a number of nutrients are continuously supplied tothe culture, right from the beginning of the fermentation or after theculture has reached a certain age, or when the nutrient(s) which are fedwere exhausted from the culture fluid. A variant of the extended batchof fed-batch, type fermentation is the repeated fed-batch orfill-and-draw fermentation, where part of the contents of the fermenteris removed at some time, for instance when the fermenter is full, whilefeeding of a nutrient is continued. In this way a fermentation can beextended for a longer time.

Another type of fermentation, the continuous fermentation or chemostatculture, uses continuous feeding of a complete medium, while culturefluid is continuously or semi-continuously withdrawn in such a way thatthe volume of the broth in the fermenter remains approximately constant.A continuous fermentation can in principle be maintained for an infinitetime.

In a batch fermentation an organism grows until one of the essentialnutrients in the medium becomes exhausted, or until fermentationconditions become unfavorable (e.g. the pH decreases to a valueinhibitory for microbial growth). In fed-batch fermentations measuresare normally taken to maintain favorable growth conditions, e.g. byusing pH control, and exhaustion of one or more essential nutrients isprevented by feeding these nutrient(s) to the culture. The microorganismwill continue to grow, at a growth rate dictated by the rate of nutrientfeed. Generally a single nutrient, very often the carbon source, willbecome limiting for growth. The same principle applies for a continuousfermentation, usually one nutrient in the medium feed is limiting, allother nutrients are in excess. The limiting nutrient will be present inthe culture fluid at a very low concentration, often unmeasurably low.Different types of nutrient limitation can be employed. Carbon sourcelimitation is most often used. Other examples are limitation by thenitrogen source, limitation by oxygen, limitation by a specific nutrientsuch as a vitamin or an amino acid (in case the microorganism isauxotrophic for such a compound), limitation by sulphur and limitationby phosphorous.

The amino acid may be recovered by any method known in the art.Exemplary procedures are provided in the following: Van Walsem, H. J. &Thompson, M. C., J. Biotechnol. 59: 127-132 (1997), and U.S. Pat. No.3,565,951, both of which are incorporated herein by reference.

All patents and publications referred to herein are expresslyincorporated by reference.

EXAMPLES Example 1 Chemical Mutagenesis and Selection of ValineAuxotrophs

A lysine producing Corynebacterium strain BF 100 was mutagenized with analkylating agent as described in Miller, J. H. 1972 (Miller, J. H. 1972Experiments in Molecular Genetics. Cold Spring Harbor Laboratory).Colonies were replica plated onto minimal medium (MM). Those that didnot grow on MM but grew on complete medium (CM) were identified asauxotrophs. Those auxotrophs that were capable of growth on MM whensupplemented with L-valine and L-isoleucine were selected for lysineyield analysis.

MM consisted of 20 g D-glucose, 10 g ammonium sulfate, 2.5 g urea, 1 gKH2PO4, 0.4 g MgSO4.7H20, 1 g NaCl, 0.01 g MnSO4.H_(20, 0.01) gFeSO4.7H20, 10 mg pantothenate, 50 mg biotin, 200 mg thiamine, and 50 mgniacinamide per liter at pH 7.2. When L-amino acids were used tosupplement MM, 50 mg/L of each was used. MMIV was MM with isoleucine andvaline added.

The growth pattern of a parent strain and a high yield-derivativeproduced by chemical mutagenesis on minimal agar plates supplementedwith three amino acids is presented in Table 1. Supplements are at 50mg/L L-amino acids. Growth is presented as relative colony size after 3days at 30 C.

Example 2 Production of Branched Chain Auxotrophs with rDNATechnology 1. Preparation of a Deleted ilvB Gene

The ilvBN operon of Corynebacterium lactofermentum (ATCC 21799) wasamplified by PCR and cloned into pCR-Script to make pAL203. The ilvBgene contains a 390 bp region separated by 2 EcoNI restriction sites.EcoNI does not cut the plasmid pCR-Script. The ilvB deletion allele wasdesigned by cutting the plasmid pAL203 with EcoNI followed byselfligation to yield pAL203delta.

2. Homologous Recombination of a Modified ilvBN Allele into theCorynebacterium Chromosome

A vector for allele exchange by double crossover was constructed asdescribed by Maloy et al. 1996 (Maloy S. R., Stewart V. J., and TaylorR. K. 1996 Genetic Analysis of Pathogenic Bacteria: A Laboratory Manual,Cold Spring Harbor Press). ATCC 37766 was the source of pK184 a plasmidthat replicates in E. coli but not in Corynebacterium. A sacB gene wassubcloned into its SspI site to give pJC3. pJC3 cannot replicate inCorynebacterium. Any kanamycin resistant colonies will have the vectorintegrated into the chromosome by homologous recombination at a sitewithin the cloned gene. Lethal expression of the sacB gene on theintegrated vector prevents growth in the presence of sucrose. Growth inthe presence of sucrose requires a second cross over to occur along anhomologous region of the cloned insert. If the first and secondcrossovers flank a modification (deletion, site mutation), then themodified allele of ilvBN will be exchanged for the allele present on thechromosome of the host strain.

The modified allele of the ilvBN operon from pAL203Δ was subcloned intothe integration vector pJC3 and electroporated into the BF100 strain ofCorynebacterium and plated on rich medium plates lacking sucrose buthaving kanamycin (DMK). Colonies were picked and grown in rich brothlacking sucrose and kanamycin for 48 hrs. Cultures were streaked ontorich plates lacking kanamycin but having sucrose. Single colonies werepicked from sucrose plates and replica plated on DMK, SM1, MM and MMIV.Strains that had no kanamycin resistance, could grow on sucrose, andcould not grow on MM but could grow on MMIV were selected for shakeflask experiments. LC10 is a BF 100-derived auxotroph.

The growth pattern of a parent strain and a high yield-derivativeproduced with recombinant DNA methods on a series of minimal agar platessupplemented with three amino acids is presented in Table 2. Supplementsare at 50 mg/L L-amino acids. Growth is presented as relative colonysize after 3 days at 30 C.

Example 3 Shake Flask Determination of L-Lysine Yield from ValineAuxotroph Strain Produced by Random Chemical Mutagenesis

B4B inoculum was prepared by picking a single colony from an S1 plateand transferring to S1 broth. S1 was made by combining 50 g sucrose, 3 gK2HP04, 3 g urea, 0.5 g MgSO4.7H_(20, 20) g polypeptone, 5 g beefextract, 0.9 mg D-biotin, 3 mg thiamine, 125 mg niacinamide, 0.5 gL-methionine, 0.25 g L-threonine, 0.5 g Lalanine per liter of water andadjusting the pH to 7.3. Plates included 20 g/L agar. After 16 hr growthof cultures in S1 broth, an equal volume of 30% glycerol was added andcultures were frozen at −80 C.

Baffled 250 mL seed shake flasks with 20 mL of SFM were inoculated with0.1 mL of thawed inoculum. Seed medium (SFM) consisted of 60 gD-glucose, 3 g K2HP04, 3 g urea, 0.5 g MgSO4.7H20, 20 g polypeptone, 5 gbeef extract, 3 mg D-biotin, 125 mg niacinamide, 0.5 g L-methionine,0.25 g L threonine, and 0.5 g L-alanine per liter of water with pHadjusted to 7.3. Cultures were grown at 30 C for 16 hrs and aerated at240 rpm with a 2 inch displacement. Two mL of seed culture was used toinoculate 21 mL of fermentation medium (FM4). FM4 medium was made bymixing 16 mL of main medium with 5 mL of dextrose stock. Dextrose stockwas 180 g D-glucose plus 500 mL water. Main medium contained 0.083 gMnSO4, 0.4 mg D-biotin, cornsteep liquor, raffinate and 50 g CaC03 perliter. Cornsteep was added so that the final volume of FM4 was 4% drysolids. Raffinate was added so that the final volume of FM4 had 5%ammonium sulfate. Cultures were grown for 48 hrs at 30 C in 250 mLbaffled shake flasks and aerated at 240 rpms with a 2 inch displacement.

Table 3 presents data on the production of L-lysine in shake flasks byCorynebacterium strain improved by selection for valine and isoleucinerequirement.

Example 4 Shake Flask Determination of L-Lysine Yield from ValineAuxotroph Strain Produced by rDNA Methodology

Table 4 presents data on the production of L-lysine in shake flasks byCorynebacterium strains improved by deleting 390 bases of DNA sequencefrom the chromosomal copy of the ilvBN operon (see Example 2). Cultureswere grown and analyzed as described in Example 3.

Example 5 Microfermenation Determination of Lysine Yield by ValineAuxotroph

Inoculum was grown in 500 mL S1 in a 2 L baffled shake flask for 18 hrs.3.1 L of FM4 medium was used in 4 L microfermentors. Temperature and pHwere maintained at 32 C and 7.2, respectively. Agitation was increasedfrom 700 rpms to 950 rpms at 20 hrs. Air was fed at 4.5 LPM. Dextrosewas maintained at 3 g/L. Fermentation time was 48 hrs.

Table 5 presents data on the production of L-lysine in 4 literfermentors using strains of Corynebacterium which cannot synthesizeL-isoleucine and L-valine.

Example 6 L-Lysine Production by Bradytroph Produced by In VivoMutagenesis of Cloned IL VBN 1. Preparation of a Defective ilvBN Operonthat Produces a Functional AHAS Enzyme

The il vBN operon of pAL203 was subcloned into the shuttle vector pM2 togive pVAL1, pM2 can replicate in both E. coli and Corynebacterium. pVal1was transformed into the mutagenic strain XL1RED from the Stratagene Co.Mutagenized plasmid was prepared according to the XL1RED kitinstructions and electroporated into a valine auxotroph,Corynebacterium. A valine auxotroph is unable to grow on MM plateswithout supplementation by isoleucine and valine or geneticcomplementation with a functional ilvBN operon.

Kanamycin resistant transformants were selected from S1 plates andreplica plated on to MM plates. Those colonies that grew on MM platesshowed functional complementation of the ilvB deletion. Colonies thatwere smaller than the colonies of the valine auxotroph with the parentplasmid (PVAL1) were selected for the valine auxotroph activity assays,pRV1B5 is a plasmid derived from pVAL1 that can replicate in E. coli andCorynebacterium. In the valine auxotroph strain, it produced AHASactivity at less than 1% of the specific activity of AHAS produced bypVAL1. The ilvBN operon of this construct has leaky AHAS activity.

2. Homologous Recombination into Corynebacterium Chromosomal DNA

The RV1B5 leaky allele of the ilvBN operon was subcloned into theintegration vector pJC3 and used to exchange the leaky allele for thedeletion allele in a valine auxotroph by homologous recombination asdone in Example 2. BF100-1030 is a valine bradytroph constructed withthe RV1B5 allele. Table 6 presents data showing that BF100-1030 makesless valine in shake flasks than its parent strain. Table 7 shows thatBF100-1030 bradytroph has improved growth over the auxotrophs in Table5. Table 7 also shows that the bradytroph produces less valine inmicrofermentors than the parent strain.

Tables

Data presented in the following tables are discussed in the Examplessection. Note that the term “Growth” refers to the optical densitymeasured at 660 nm; the term “Titre” refers to the grams of amino acidper liter; the term “Yield” is defined by the following formula: [(glysine/L/(g dextrose consumed/L)]*100; B4B=a valine autotrophconstructed with chemical mutagenesis; LC10=a valine autotrophconstructed by replacing the chromosomal ilvB gene with the ilvBdeletion allele of pAL203 A; BF 100-1030=a valine bradytroph constructedby replacing the chromosomal ilvB gene with the RV1B5 leaky allele.TABLE 1 Valine Auxotroph Selection Following Chemical Mutagenesis AminoAcid Supplement Relative Growth Agar Plate ile leu val BF100 B4B MM − −− 5 0 MM + + − 5 0 MM + − − 5 2 MM − + + 5 0 MM + + + 5 5

TABLE 2 Auxotroph Selection Following rDNA Modification Amino AcidSupplement Relative Growth Agar Plate ile leu val BF100 LC10 MM − − − 30 MM + + − 3 0 MM + − + 3 1 MM − + + 3 0 MM + + + 3 3

TABLE 3 Shake Flask Determination of L-lysine Yield From a ValineAuxotroph Strain Produced by Random Chemical Mutagenesos Strain GrowthLysine Titre % Yield Parent-1 33 18 32 B4B 33 17 44

TABLE 4 Shake Flask Determination of L-lysine Yield From a ValineAuxotroph Strain Produced by rDNA Methodology Strain Growth Lysine Titre% Yield BF100 35 25 38 LC10 35 28 43

TABLE 5 Microfermentation Determination of Lysine Yield Strain GrowthLysine Titre % Yield Valine Titre BF100 90 113 37 8.9 LC10 63 86 50 1.1B4B 70 91 51 —

TABLE 6 Shake Flask Determination of Decreased L-Valine Titre from aValine Bradytroph Strain Produced by Integrating the RV1B5 allele ifilvB into the Chromosome Strain Growth Lysine Titre % Yield Valine TitreBF100 36 27 29 5.2 BF100-1030 42 22 25 3.5

TABLE 7 Microfermentation Determination of Decreased L-Valine Titre froma Valine Bradytroph Strain Produced by Integrating the RV1B5 Allele ofilvV into the Chromosome Strain Growth Lysine Titre % Yield Valine TitreBF100 89 134 43 9 BF100-1030 78 123 44 6

1-24. (canceled)
 25. A method of making a microorganism that producesamino acid X comprising: (a) selecting a parental population ofmicroorganisms A that contain an ilvBN operon and produce said aminoacid from dextrose in percent yield Y; (b) mutagenizing said parentalpopulation of microorganisms A to produce a population of mutagenizedmicroorganisms B; (c) selecting from the population of mutagenizedmicroorganisms B, at least one mutagenized microorganism C that has atleast one of a deletion, an insertion, and a point mutation in saidilvBN operon and that produces said amino acid X from dextrose inpercent yield Z, wherein said percent yield Z is greater than saidpercent yield Y, and wherein said amino acid X is selected from thegroup consisting of L-lysine, L-threonine, L-methionine, and homoserine.26. The method of claim 25, wherein said parental population ofmicroorganisms A is mutagenized by random chemical mutagenesis.
 27. Themethod of claim 26, wherein said microorganism C comprises an endogenousilvBN operon.
 28. The method of claim 25, wherein microorganism A ismutagenized by rDNA mutagenesis of the ilvBN operon of microorganism A.29. The method of claim 28, wherein the mutagenesis is the result of atransformation with a vector comprising a recombinant polynucleotidecomprising SEQ ID NO:
 5. 30. The method of claim 25, wherein saidmutagenized microorganism C is bradytrophic for valine.
 31. The methodof claim 30, wherein said mutagenized microorganism C is bradytrophicfor valine and isoleucine.
 32. The method of claim 30, wherein saidmutagenized microorganism C is bradytrophic for valine and leucine. 33.The method of claim 32, wherein said mutagenized microorganism C isbradytrophic for valine, leucine, and isoleucine.
 34. The method ofclaim 25, wherein said parental microorganism A, said microorganism B,and said mutagenized microorganism C are strains of Corynebacterium. 35.A bacterial culture medium comprising a microorganism made by a methodof claim
 25. 36. A microorganism C obtained by the method of claim 25.37. A microorganism according to claim 1, wherein said mutagenizedmicroorganism C produces amino acid X in a yield at least 1% greaterthan the yield produced by parental microorganism A when mutagenizedmicroorganism C and parental microorganism A are cultured in identicalmedia under identical conditions.
 38. A microorganism according to claim37, wherein said identical conditions occur in a microfermentation. 39.A strain of Corynebacterium comprising an ilvBN operon comprising thenucleotide sequence of SEQ ID NO: 5.